Anthrax toxin, produced by Bacillus anthracis, is composed of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF) (Leppla, Handbook of Natural Toxins 8:543–572 (Moss et al., eds., 1995)). PA alone has no toxic effect upon cells, but instead binds to specific cell surface receptors. Upon proteolytic activation to a 63-kDa fragment (PA63), PA forms a heptameric membrane-inserted channel, which mediates the entry of EF and LF into the cytosol via the endosomal pathway (Gordon et al., Infect. Immun. 56:1066–1069 (1988); Milne et al., J. Biol Chem. 269:20607–20612 (1994)). Thus, EF or LF are toxic to cells when combined with PA.
EF is an adenylate cyclase, and together with PA forms a toxin referred to as edema toxin (Leppla, Proc. Natl. Acad. Sci. USA 79:3162–3166 (1982)). LF and PA together form a toxin referred to as lethal toxin (“LT”). Until the present discovery, however, the specific activity of LP in the cell was unknown. Lethal toxin is the dominant virulence factor produced by B. anthracis and is the major cause of death of infected animals (Pezard et al., Infect. Immun. 59:3472–3477 (1991)). Intravenous injection of lethal toxin causes death of Fisher 344 rats in as little as 38 minutes (Ezzell et al., Infect Immun. 45:761–767 (1984)), and incubation in vitro with mouse macrophages causes lysis in 90–120 minutes (Friediander, J. Biol Chem. 261:7123–7126 (1986)).
LF contains a limited sequence homology to a putative zinc-binding site at residues 686–690, HEFGH, characteristic of metalloproteases (Klimpel et al., Mol. Microbiol. 13:1093–1100 (1994)). Substitution of the H or E residues inactivates LF (e.g., as in the recombinant LF mutant E687C) (Klimpel et al., 1994, supra) and decreases its binding of zinc (Klimpel et al., 1994, supra; Kocki el al. FEMS Microbiol. Lett. 124:343–348 (1994)). Certain metalloprotease inhibitors also protect macrophages against lethal toxin. (Klimpel et al., 1994, supra; Menard et al., Biochem J. 320:687–691 (1996)). However, no physiological substrate has been identified for LF, and LF protease activity has not been demonstrated.